1. Field of the Invention
The present invention relates to the determination, detection and quantification, in a biological sample, of a gliotoxic factor such as associated with multiple sclerosis.
2. State of the Art
A "biological sample" is understood, in particular, as being a specimen of the biological fluid type, live tissue or tissue fragment, mucus sample, organ or organ fragment type, or any culture supernatant obtained with the aid of an aforementioned specimen.
In accordance with document WO-A-95/21859, which is an application filed in the name of the applicant, a factor which is cytotoxic for glial cells (astrocytes, oligodendrocytes and microgliocytes), and which is termed gliotoxic factor below, has been isolated and/or characterized. While this factor is, in particular, associated with multiple sclerosis, it could also be associated with other neurodegenerative or autoimmune diseases.
In the absence of a complete sequence for this gliotoxic factor, the latter can be characterized either using a process (among others) which enables the factor to be isolated or purified or using different biological, biochemical or chemical properties or characteristics.
According to document WO-A-95/21859, this gliotoxic factor is characterized by all the following properties, considered separately or in combination:
it possesses a toxic activity for human or animal astrocytic cells, with its effect being that of eliciting cytomorphological disruption of their network of intermediate filaments and/or degradation of the proteins of these intermediate filaments and/or cell death, in particular by apoptosis, PA1 its activity is associated with at least one glycoprotein, PA1 this gliotoxic factor consists in the main, if not entirely, of a light-weight fraction which is centred on an apparent molecular weight of approximately 17 kD; this light-weight fraction is resistant, under standard non-denaturing conditions, to the hydrolytic action of pronase or trypsin or proteinase K; and this same light-weight fraction exhibits strong affinity for lectins, in particular concanavalin A. PA1 a starting fraction of this sample, which is, where appropriate, enriched in the said gliotoxic factor by any appropriate prior treatment, is to hand, PA1 this starting fraction is incubated with a reference culture medium which, for example, comprises glial cells, in particular immortalized cells, for example astrocytic cells, and PA1 the dead or living glial cells are detected and/or quantified using any appropriate technique, for example using a calorimetric assay employing calcein AM and homodimeric ethidium, respectively. PA1 a process which is able to comprise a "non-invasive" specimen, PA1 a process which is simple to implement, which is sensitive and which is specific for the gliotoxic factor. PA1 fragmentation of the cellular DNA of the apoptotic cells; PA1 preservation of the integrity of the cytoplasmic membrane of the apoptotic cells; PA1 alterations to the structure and size of the apoptotic cells. PA1 the ploidy of the macroglial cells, that is to say determine the quantity of DNA remaining in the cells after extraction of the DNA fragments, it being understood that the DNA quantity is preserved in non-apoptotic cells since there is no fragmentation; on the basis of the ploidy, it is also possible to study the cell cycle; PA1 the state of the macroglial cells; thus, cell morphology is determined by the size of the cells, for example using the so-called FALS (forward light angle scatter) technique, since the death of astrocytes by apoptosis brings about a decrease in their size; by the structure of the said cells, for example using the so-called Ss (side scatter) technique; and, where appropriate, by the presence and/or quantity of a protein, such as the glial fibrillar acid protein (GFAP), which makes it possible to discriminate between the cell subpopulations, for example using a labeled anti-GFAP antibody which can be identified directly or indirectly. PA1 detaching the adherent macroglial cells, PA1 treating the cells which have thus been detached with an agent for cell fixation and permeabilizing the cytoplasmic membrane of the said cells, PA1 extracting intracellular DNA fragments resulting from the apoptosis, PA1 labeling the remaining cellular DNA with an appropriate label, and PA1 detecting, by flow cytometry, the ploidy of the macroglial cells. PA1 inducing a necrosis, in particular by incubation, in a sample of living macroglial cells which is different from the sample to be analyzed, PA1 labeling the DNA debris which result from the necrosis, with the said debris being associated with fragments of the cytoplasmic membrane of the necrotic macroglial cells, and with the labeling being effected after detaching the said cells, PA1 locating, in flow cytometry, the cells which have died by necrosis and appropriately adjusting the cytometer, thereby enabling the cells which have died by necrosis to be excluded during the step of detecting the cells which have died by apoptosis, PA1 detecting the cells which have died by apoptosis. PA1 dividing the cells of the biological sample into two equal parts and detaching the cells, PA1 detecting, by flow cytometry, in one of the two parts, the macroglial cells which have died by apoptosis or by necrosis, on the one hand, and/or the living macroglial cells, on the other hand, in which step the said fraction is treated with an agent for cell fixation and for permeabilization of the cytoplasmic membrane; the DNA fragments are extracted; the intracellular DNA is labeled with a label and the remaining intracellular DNA is detected, PA1 detecting, by flow cytometry after fixation without extraction, in the other of the two parts, the living macroglial cells and the apoptotic macroglial cells, on the one hand, and/or the necrotic macroglial cells, on the other hand, PA1 deducing, from the detections effected on each of the two parts, the quantity of apoptotic macroglial cells.
Consequently, the detection and/or quantification of this gliotoxic factor in any biological sample is a worthwhile and effective analytical tool, in particular for the diagnosis of different pathologies, including multiple sclerosis, and the prediction, monitoring and therapy of this disease. Still in accordance with document WO-A-95/21859, the content and description of which are hereby incorporated into the present patent application by reference, a process for detecting and/or quantifying this gliotoxic factor in a biological sample, for example a sample of cerebrospinal fluid, has been described and proposed. Such a process comprises at least the following treatments:
Such a process, which is implemented for detecting and monitoring the therapy of multiple sclerosis, presupposes the existence of a so-called "invasive" specimen, for example of cerebrospinal fluid, that is to say which requires a prior medical or surgical act.